Impaired Anticoagulant Response to Infusion of Thrombin in Atherosclerotic Monkeys Associated With Acquired Defects in the Protein C System

Abstract

Abstract-To examine the effects of atherosclerosis on the protein C anticoagulant pathway in vivo, we measured anticoagulant responses to intravenous administration of human ␣-thrombin or activated protein C (APC) in cynomolgus monkeys. Two groups of monkeys were fed either a control diet (nϭ18) or an atherogenic diet (nϭ12) that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. A third group (nϭ8) was fed an atherogenic diet for 15 months, and then fed the atherogenic diet supplemented with B vitamins for 6 months to correct the hyperhomocyst(e)inemia. The plasma homocyst(e)ine level was higher in monkeys fed the atherogenic diet (9.6Ϯ1.0 mol/L) than in monkeys fed the control diet (3.7Ϯ0.2 mol/L) or the atherogenic diet with B vitamins (3.6Ϯ0.2 mol/L) (PϽ0.001). Infusion of thrombin produced a much greater prolongation of the activated partial thromboplastin time in monkeys fed the control diet (52Ϯ10 seconds) than in monkeys fed the atherogenic diet either with (24Ϯ4 seconds) or without (27Ϯ5 seconds) supplemental B vitamins (PϽ0.02). Thrombin-dependent generation of circulating APC was higher in control (294Ϯ17 U/mL) than in atherosclerotic (240Ϯ14 U/mL) monkeys (PϽ0.05), although levels of fibrinogen, plasminogen, D-dimer, and thrombin-antithrombin complexes were similar in each group. Injection of human APC produced a similar prolongation of the activated partial thromboplastin time in control (31Ϯ3 seconds) and atherosclerotic (29Ϯ2 seconds) monkeys. These findings provide evidence for impaired anticoagulation, due partly to decreased formation of APC, in atherosclerosis. Key Words: atherosclerosis Ⅲ cholesterol Ⅲ homocysteine Ⅲ protein C Ⅲ thrombomodulin T hrombin, a major regulator of hemostasis, has both procoagulant and anticoagulant properties. 1 Procoagulant effects of thrombin include proteolytic activation of factors V, VIII, and XI; cleavage of fibrinogen to form a fibrin clot; and stimulation of platelet aggregation. A major anticoagulant effect of thrombin involves activation of protein C, a vitamin K-dependent plasma anticoagulant. Thrombin's procoagulant and anticoagulant activities are regulated by thrombomodulin, a cofactor that is expressed on the luminal surface of vascular endothelium. 2 When bound to thrombomodulin, thrombin's ability to catalyze procoagulant reactions is inhibited, but its ability to activate protein C is enhanced Ͼ1000-fold. 3 The protein C anticoagulant pathway is impaired in patients with inherited deficiencies of protein C or protein S and also in patients with a mutation in factor V (factor V Leiden) that renders it resistant to activated protein C (APC). 4,5 The physiological importance of the protein C anticoagulant pathway is underscored by the observation that resistance to APC is found in 20% to 50% of patients with venous thromboembolism. 6 Resistance to APC that is independent of factor V Leiden may be associated with increased risk of stroke. 7,8 In a previous study, we observed impaired endotheliumdependent vasodilatation and decreas...

Platelet factor 4 enhances generation of activated protein C in vitro and in vivo

“…We also performed studies in cynomolgus monkeys, using as our experimental model low-dose infusion of thrombin, a potent stimulus to generation of APC that produces large increases in circulating APC levels accompanied by a pronounced systemic anticoagulant effect. [16][17][18] Our results show that PF4 enhances the protein C-activating cofactor activity of TM both in cultured endothelial cells in vitro and in intact vasculature in vivo.…”

“…On each of the 2 study days, the animals were sedated with ketamine hydrochloride (20 mg/kg, administered intramuscularly) and anesthetized with sodium pentobarbital (20 mg/kg, administered intravenously) as described previously. 18 A nonobstructive catheter was inserted into an axillary artery for blood sampling, and the axillary vein was cannulated for administration of PF4, thrombin, and supplemental anesthesia (sodium pentobarbital, 5 mg/kg/h). Arterial pressure was monitored continuously.…”

“…Immediately following the infusion of PF4 or vehicle, human ␣-thrombin (1.0 g/kg; Enzyme Research Laboratories) was infused over 10 minutes as described previously. 18 Blood was collected from the axillary artery catheter before and after infusion of PF4 or vehicle, and at intervals (0-120 minutes) after infusion of thrombin, directly into a 1/10 volume of 3.8% sodium citrate with 30 mM benzamidine (for determination of APC) or 3.8% sodium citrate without benzamidine (for other hemostasis assays). Blood samples were immediately placed on ice and plasma was isolated by centrifugation at 2500g for 30 minutes at 4°C.…”

“…18,24 The assay was standardized using a pool of normal monkey plasma that contained approximately 6.5 ng/mL APC.…”

Mikamo Lecture-A Radical View of the 'Superfamily' of Cardiovascular Risk Factors-

“…We observed both endothelial vasomotor dysfunction and an acquired defect in the protein C anticoagulant system in the monkeys. 36 Impairment of anticoagulation may contribute to the propensity for thrombosis in atherosclerotic arteries.…”